Stabilization of pleuromutilin derivatives against oxidation by sodium hypochlorite in aqueous solution

ABSTRACT

A composition and method which provides a compound of the formula I: ##STR1## wherein either R 1  is ethyl or vinyl. n is an integer from 2 to 5 and each of R 2  and R 3  is alkyl of 1 to 10 carbon atoms. 
     Also provided is a hypochlorite scavenger in solution with the formula I compound which stabilizes the compound against oxidation by sodium hypochlorite. 
     A preferred solution contains the compound of formula I wherein R 1  is vinyl, R 2  and R 3  are ethyl as a salt of hydrogen fumarate i.e. tiamulin hydrogen fumarate. The solution is added to drinking water which is fed to farm animals as treatment for mycoplasma infection.

BACKGROUND OF THE INVENTION

The sterilization of drinking water pipelines and utensils for farmanimals is sometimes carried out using a solution of sodiumhypochlorite. There are also occasions in which pond water is similarlysterilized for animal consumption in situations where water supply mainsare ot available.

Baughn discloses in U.S. Pat. No. 3,987,194 issued Oct. 19, 1976 thatoctahydro-5,8-dihydroxy-4,6,9,10-tetramethyl-6-vinyl-3a,9-propano-3aH-cyclopentacycloocten-1(4H)-one,8[[2-(diethylamino)ethyl]thio]acetate;oroctahydro-5,8-dihydroxy-4,6,9,10-tetramethyl-6-vinyl-3a,9-propano-3aH-cyclopentacycloocten-1(4H)-one,8-[[2-[4-(2-hydroxyethyl)-1-piperazinyl]ethyl]thio]acetateis useful against swine dysentery. And that the agents can be added tothe drinking water.

U.S. Pat. No. 3,919,290 issued Nov. 11, 1975, discloses various14-deoxymutilins, which are pleuromutilin derivatives. The compounds aredisclosed as being useful because of their pharmacological activity inanimals, and more specifically, they are said to be useful asantibiotics with antibacterial action. The reference discloses thecompounds to be useful both prophylactically and therapeutically indomestic animals, particularly pigs and poultry.

14-Deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mutilin, andpharmaceutically acceptable salts thereof, is disclosed by thereference. The base compound has the non-proprietary name tiamulin.

OBJECTS OF THE INVENTION

It has been observed that residues of sodium hypochlorite in drinkingwater can adversely affect the therapeutic activity of tiamulin hydrogenfumarate by imparting upon the antibiotic an oxidative decomposition tothe corresponding sulphoxide.

It is an object of the invention to provide a means of removing theadverse effects of sodium hypochlorite by the use of selectedscavengers.

These scavengers are chosen such that they can either be added to thedrinking water prior to the incorporation of the antibiotic or they canbe added with the antibiotic in which case they are able topreferentially react with the sodium hypochlorite.

It is an object of the invention to provide a treatment for mycoplasmainfection in farm animals.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention there is provided a compound of theformula I: ##STR2## wherein either R₁ is ethyl or vinyl.

n is an integer from 2 to 5 and each of R₂ and R₃ is alkyl of 1 to 10carbon atoms.

Also provided is a hypochlorite scavenger in solution with the formula Icompound.

A preferred solution contains the compound of formula I wherein R₁ isvinyl, R₂ and R₃ are ethyl as a salt of hydrogen fumarate i.e. tiamulinhydrogen fumarate.

There is interaction between the compound of formula I, for exampleOctahydro-5,8-dihydroxy-4,6,9,10-tetramethyl-6-vinyl-3a,9-propano-3aH-cyclopentacycloocten-1(4H)-one,8-[[[2-(diethylamino)ethyl]thio]acetate], fumarate (1:1) hereinaftercalled tiamulin hydrogen fumarate, and sodium hypochlorite which isquantitative and spontaneous at room temperature. The degradationproducts resulting from the interaction are the sulphoxide and thehydrolysis products of the compound of formula I. The former is presentin considerable quantities, whereas the latter is only a minorcomponent.

The compounds of formula I can be added in concentrations rangingbetween 0.0045% by weight and 0.0125% by weight to the drinking water offarm animals for the treatment of mycoplasma infections. It is renderedunstable if residues of the sodium hypochlorite sterilant are present inquantities exceeding 1 ppm. of available chlorine. The reaction isinstantaneous and primarily oxidative in nature, the resultingdecomposition product being the corresponding sulphoxide. A minor amountof hydrolysis product is also produced.

The extent of the decomposition is dependent on the available chlorinecontent of the water. This is illustrated by the data given in Tables 1Aand 1B which show the effect of various concentrations of availablechlorine on the decompositions of (a) 0.01% tiamulin base in 50% aqueousmethanol (to solubilize the base) and (b) 0.0125% tiamulin hydrogenfumarate (with 0.0153% lactose B.P.) in water. Thus, the reaction isshown to be quantitative in the presence of sufficient quantities ofavailable chlorine.

                  TABLE 1A                                                        ______________________________________                                        % Recovery of   PPM available                                                 tiamulin base   chlorine                                                      (HPLC ASSAY)    concentration                                                 ______________________________________                                        93              2.5                                                           85              5                                                             80              7.2                                                           76              10                                                            70              12                                                            57              18                                                            34              24                                                            23              30                                                            ______________________________________                                    

                  TABLE 1B                                                        ______________________________________                                                           PPM                                                        % recovery of tiamulin                                                                           available                                                  hydrogen fumarate  chlorine                                                   ______________________________________                                        92 (HPLC)          1                                                          85 (Micro)         1                                                          88 (HPLC)          2                                                          90 (Micro)         2                                                          74 (HPLC)          3                                                          76 (Micro)         3                                                          66 (HPLC)          10                                                         68 (Micro)         10                                                          6 (HPLC)          25                                                         12 (Micro)         25                                                         ______________________________________                                    

Sodium hypochlorite is a well known oxidizing agent which is prepared bythe interaction of chlorine gas with aqueous sodium hydroxide solution.The scavenging of sodium hypochlorite to prevent its reaction with theformula I compound can be achieved by the use of suitable reducingagents such as sodium thiosulphate, sodium sulphite, sodium nitrite,acetone sodium bisulphite, ascorbic acid, dithiothreitol,dithioerythritol and urea. Of these materials sodium thiosulphate,acetone sodium bisulphite, ascorbic acid, dithiothreitol anddithioerythritol are able to preferentially react with sodiumhypochlorite when added with the the formula I compound. The remainingmaterials are only suitable when added prior to the addition of theformula I compound.

The suitability of the latter named scavengers depends on theirconcentration relative to that of the residual sodium hypochlorite.Thus, their scavenging performances can be gauged by the informatonsummarised in Tables 1 to 3 which represent the effect of variousconcentrations of sodium thiosulphate, acetone sodium bisulphite, andascorbic acid when added with 0.0125% tiamulin hydrogen fumarate todrinking water containing excessive residues of sodium hypochlorite(i.e. 5 ppm. available chlorine). The data clearly shows that theantibiotic activity is maintained at scavenger concentrations in excessof 40 ppm.

In practice the scavenging of residual sodium hypochlorite must beachieved in sterilized pond water for drinking or in pipelines andutensils through which fresh, unsterilized drinking water may besupplied. These requirements may be achieved in several ways, as forexample:

(1) The scavenger and the pleuromutilin compound formulation aredissolved together in a small quantity of sterilized or unsterilizedwater and the resulting concentrate is the added to the bulk of thesterilized drinking water giving a supply which contains effectiveantibiotic activity.

(2) The scavenger is dissolved directly in the bulk of the sterilizeddrinking water either prior to or with the requisite quantity of thepleuromutilin compound formulation, producing a supply which containseffective antibiotic activity.

(3) The scavenger and pleuromutilin compound formulation are dissolvedtogether in a small quantity of fresh water and the resultingconcentrate is added to the bulk of the fresh, unsterilized drinkingwater which when fed through the sterilized pipelines and utensils givesa supply containing the theoretical amount of effective antibioticactivity.

The following examples illustrate the invention.

EXAMPLE 1

Provide 20 liters of pond drinking water containing

(a) 0.0125% Tiamulin hydrogen fumarate

(b) 0.0153% Lactose

(c) 0.004% Sodium thiosulphate

(c) and (a) are prepared as follows:

0.8 g. of sodium thiosulphate was dissolved in 250 ml. of sterilizedpond water.

5.56 g. of tiamulin hydrogen fumarate was added and stirred untildissolved. The resulting concentrates was added to the bulk of thesterilized pond water to give a 20 liter drinking water supplycontaining effective antibiotic activity.

EXAMPLE 2

The same method as Example 1 but containing 0.004% of any of thefollowing scavengers: acetone sodium bisulphite, ascorbic acid,dithiothreitol or dithioerythritol.

EXAMPLE 3

The method of Example 1 but in which the sodium thiosulphate andtiamulin hydrogen fumarate are added simultaneously.

EXAMPLE 4

The same as for Example 3 but in which the scavenger is acetone sodiumbisulphite, ascorbic acid, dithiothreitol or dithioerythritol.

EXAMPLE 5

The same as for Examples 1-4 but in which the drinking water is freshmains, i.e. city water, for supply to a sterilized delivery system.

EXAMPLE 6

The same as for Examples 1-5 but in which the scavengers are at aconcentration of 0.0125%.

EXAMPLE 7

The same as for Examples 1-6 but in which the tiamulin hydrogen fumarateis at a concentration of 0.0045% and the lactose is at a concentrationof 0.0255%.

EXAMPLE 8

Provide 20 liters of pond drinking water containing

(a) 0.0125% Tiamulin hydrogen fumarate

(b) 0.0153% Lactose

(c) 0.004% Sodium thiosulphate

(c) and (a) are prepared as follows:

0.8 g. of sodium thiosulphate was dissolved, with stirring, in 20 litersof sterilized pond water.

5.56 g. of tiamulin hydrogen fumarate was then added, with stirring, togive a supply containing effective antibiotic activity.

EXAMPLE 9

The same as for Example 8, but in which the scavenger is acetone sodiumbisulphite, ascorbic acid, dithiothreitol, dithioerythritol, sodiumbisulphite, sodium nitrite or urea, or a combination of any of these.

                  TABLE 1                                                         ______________________________________                                        Scavenging Effect of Sodium Thiosulphate                                                          % Tiamulin Hydrogen                                                           Fumarate Recovered                                        Sample                HPLC    Micro                                           ______________________________________                                        Control               100     100                                             Control + 5ppm. av. Cl.                                                                             77.3    76                                              Control + 5ppm. av. Cl. + 3ppm. Thio                                                                89.8    96                                              Control + 5ppm. av. Cl. + 8ppm. Thio                                                                92.0    89                                              Control + 5ppm. av. Cl. + 13ppm. Thio                                                               95.0    97                                              Control + 5ppm. av. Cl. + 26ppm. Thio                                                               97.2    98                                              Control + 5ppm. av. Cl. + 53ppm. Thio                                                               100.1   99                                              ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Scavenging Effect of Acetone Sodium Bisulphite (ASB)                                              % Tiamulin Hydrogen                                                           Fumarate Recovered                                        Sample                HPLC    Micro                                           ______________________________________                                        Control               100.0   100                                             Control + 5ppm. av. Cl.                                                                             77.0    83                                              Control + 5ppm. av. Cl. + 4ppm. ASB                                                                 78.8    88                                              Control + 5ppm. av. Cl. + 13ppm. ASB                                                                89.9    99                                              Control + 5ppm. av. Cl. + 21ppm. ASB                                                                94.6    107                                             Control + 5ppm. av. Cl. + 42ppm. ASB                                                                99.3    106                                             Control + 5ppm. av. Cl. + 84ppm. ASB                                                                105.0   107                                             ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Scavenging Effect of Ascorbic Acid (AA)                                                           % Tiamulin Hydrogen                                                           Fumarate Recovered                                        Sample                HPLC    Micro                                           ______________________________________                                        Control               100.0   100                                             Control + 5ppm. av. Cl.                                                                             81.2    89                                              Control + 5ppm. av. Cl. + 3ppm. AA                                                                  --      --                                              Control + 5ppm. av. Cl. + 8ppm. AA                                                                  88.0    94                                              Control + 5ppm. av. Cl. + 13ppm. AA                                                                 --      --                                              Control + 5ppm. av. Cl. + 26ppm. AA                                                                 91.2    88                                              Control + 5ppm. av. Cl. + 53ppm. AA                                                                 95.6    98                                              ______________________________________                                    

Examples 1 through 9 show hypochlorite scavenging antibiotic solutionsof the present invention. The antibiotic effectiveness is increased bythe action of the hypochlorite scavenger. By scavenging hypochloriite asubstantial portion of the antibiotic formula I compound is maintainedin its unoxidized and unhydrolized state.

Table IA shows that as the concentration of available chlorine increases(in the absence of hypochlorite scavenger) the percentage recovery oftiamulin base decreases. The available chlorine is from hypochlorite.

Table IB shows that as the concentration of available chlorine increases(in the absence of hypochlorite scavenger) the percentage recovery oftiamulin hydrogen fumarate decreases. The percent recovery of tiamulinhydrogen fumarate is measured by both high performance liquidchromatography (HPLC) and microanalysis (micro). In Table I the resultsare shown for using sodium thiosulphate to scavenge for availablechlorine. In Table II the results are shown for using acetone sodiumbisulfite to scavenge for available chlorine in the amount of five partsper million. In Table III the results are shown for using ascorbic acidto scavenge for five parts per million of available chlorine.

Table IB shows that as the concentration of available chlorine increasesthe percent recovery of tiamulin hydrogen fumarate decreases in theabsence of hypochlorite scavenger.

Comparing Tables I through III with Table IB, it is seen that when 3parts per million of available chlorine is present in the absence of ascavenger, only 74 to 76% of the tiamulin hydrogen fumarate is obtained.Whereas Table I shows that in the presence of an even higherconcentration of available chlorine (5 parts per million of availablechlorine) 99 to 100% of the tiamulin hydrogen fumarate may be coveredwhen sodium thiosulphate scavenger is present. Similarly, Table II showsthat all of the tiamulin hydrogen fumarate may be recovered when acetonesodium bisulphite is present. In Table III 95 to 98% of the tiamulinhydrogen fumarate is shown to be recovered from a solution containingfive parts per million of available chlorine when ascorbic acid ispresent in the amount of 53 parts per million. The percent recovery oftiamulin represents the percent of effective tiamulin in the solution.Thus, it also represents the percent of effective antibiotic present inthe solution.

In summary, when available chlorine is present the concentration ofunoxidized tiamulin hydrogen fumarate was reduced. The higher theconcentration of available chlorine the lower the percentage recovery oftiamulin hydrogen fumarate. The present invention uses a scavenger foravailable chlorine in solution with compounds of formula I. Thescavenger increases the effectiveness of the compound of formula I byincreasing the concentration of the compound of formula I which isneither oxidized nor hydrolized. The preferred weight ratio of formula Icompound to hypochlorite scavenger is from about 0.1:1 to about 500:1.

I claim:
 1. A composition for water treatment consisting essentially ofa hypochlorite scavenger and from 0.0045% to 0.0125% by weight of acompound of the formula ##STR3## wherein R₁ is either ethyl or vinyl; nis an integer from 2 to 5 and each of R₂ and R₃ is an alkyl of 1 to 10carbon atoms, and pharmaceutically acceptable salts of said compound,said hypochlorite scavenger being selected from the group consisting ofsodium thiosulphate, acetone, sodium bisulphite, ascorbic acid,dithiothreitol, dithioerythritol, sodium sulphite, sodium nitrate andurea, said compound being present in a weight ratio to said hypochloritescavenger of from about 0.1:1 to about 500:1.
 2. The composition ofclaim 1 further comprising providing said compound as a hydrogenfumarate salt and wherein R₁ is vinyl, R₂ and R₃ are each ethyl.
 3. Thecomposition of claim 1 wherein said composition further compriseslactose and said compound is tiamulin hydrogen fumarate.